Estrus synchronization in ewes treated with sponges impregnated
with different doses of medroxyprogesterone acetate
L. Simonetti
*, M.R. Blanco, J.C. GardoÂn
Lab. ReproduccioÂn Animal. Facultad de Ciencias Agrarias. Universidad Nacional de Lomas de Zamora. Ruta 4 Km. 2 (1836), Llavallol, Buenos Aires, Argentina
Received 27 October 1999; accepted 17 April 2000
Abstract
The objectives of this study were to determine residual and absorbed levels of medroxyprogesterone acetate (MAP) after treatment with intravaginal sponges impregnated with different doses of MAP in an ewe herd and to determine the effects of such treatments upon estrus incidence, interval to estrus onset and pregnancy rate. Polyurethane sponges impregnated with 40, 50 and 60 mg of MAP were prepared. Real amounts of progestagen on sponges were checked prior to sponge treatment insertion. During autumn 1999, 608 cyclic Merino ewes were treated with intravaginal sponges impregnated with different doses of MAP (groups I, 40; II, 50 and III, 60 mg). After 14 days, sponges were removed and residual levels of MAP (RMAP) on removed sponges were measured by spectrophotometry. Real amounts for unused sponges were 39.601.10, 44.101.06 and 59.101.26 mg for the intended doses of 40, 50 and 60 mg MAP respectively. RMAPs were different (p<0.05) among groups (I: 17.981.97 mg; II: 24.322.03 mg; III: 34.253.23 mg). Absorbed levels of MAP were not different among groups (I: 21.621.97 mg; II: 19.782.03 mg; III: 24.853.23 mg). Arti®cial insemination was performed in 16 ewes from Group I, 17 ewes from Group II and 11 ewes from Group III, 12 h after estrus onset. There were no differences among groups, neither for estrus incidence (I: 79.27%; II: 77.42%; III: 80.87%) nor interval to estrus onset (I: 55.941.87 h; II: 56.741.13 h; III: 57.701.02 h). There were also no differences among groups for pregnancy rate (I: 43.75%; II: 52.94%;
III: 45.45%). It was concluded that under similar conditions, a dose as low as 40 mg MAP could be effectively used for estrus synchronization in cyclic Merino ewes.#2000 Elsevier Science B.V. All rights reserved.
Keywords:Ewes; Estrus synchronization; Fertility; MAP; Sponges
1. Introduction
Synchronization of estrus in ewes has been per-formed with intravaginal sponges impregnated with natural progesterone (Robinson, 1967) and, more recently, with synthetic progestagens such as ¯uoro-gestone acetate (FGA; 9a-¯uoro-11û-hydroxy-17a
-acetoxy-pregne-4-ene-3.20 dione) or medroxyproges-terone acetate (MAP; 6a-methyl-17a
-acetoxy-pregne-4-ene-3.20 dione) (Quirke et al., 1981; Smith et al., 1981; Alifakiotis et al., 1982; Ainsworth and Shrestha, 1983; Oyediji et al., 1990; Crosby et al., 1991; Moses et al., 1997).
MAP-impregnated pessaries used for estrus syn-chronization in small ruminants are commercially available in a dose of 60 mg of such hormone (Rubianes et al., 1999). Crosby et al. (1991) postulated that a high level of progestagen followed by its rapid *Corresponding author. Tel.:54-11-42827896;
fax:54-11-42826263
E-mail address: 1_simonetti@hotmail.com (L. Simonetti).
withdrawal is a necessary condition for acceptable fertility at the synchronized estrus. On the other hand, there are suggestions that optimal fertility following synchronization with progestagens can be achieved with lower doses (Faure et al., 1983; Greyling et al., 1997).
In a preliminary study in ewes (Simonetti et al., 1999), the existence of a residue of MAP was found on sponges removed from ewes' vaginas after 14 days of permanence. This suggests that the amount of MAP effectively absorbed by these females is lower than the 60 mg dose presented on sponges. Moreover, the levels of MAP effectively used did not seem to affect fertility.
The objectives of this study were to determine residual and absorbed levels of MAP after treatment with intravaginal sponges impregnated with different doses of MAP (40, 50 and 60 mg) in an ewe herd and to determine the effects of treatment with intravaginal sponges impregnated with different doses of MAP (40, 50 and 60 mg) upon estrus incidence, interval to estrus onset and pregnancy rate.
2. Materials and methods
A total of 608 cyclic Merino ewes, 3±5 years of age and 40±45 kg body weight, were used in this study. The experiment was conducted during autumn 1999. The animals were managed under the same conditions on one farm. They were kept under natural ®eld conditions, having access to good quality grasses and maintained in good health. These females were randomly assigned in two experiments.
2.1. Experiment I
Females (n59) were randomly allocated to the following groups: Group I (n21): ewes were treated with polyurethane vaginal sponges impregnated with 40 mg MAP. Group II (n19): ewes were treated with polyurethane vaginal sponges impregnated with 50 mg MAP. Group III (n14): ewes were treated with polyurethane vaginal sponges impregnated with 60 mg MAP. Polyurethane sponges were prepared by the method already reported by Robinson (1965). Sponges were left in place for 14 days in all groups. At withdrawal, each pessary from all groups was
placed into a sterile ¯ask until assay in order to determine the residue (remnant MAP on removed sponges used for estrus synchronization). Some sponges impregnated with 40 (n10), 50 (n10) and 60 (n10) mg MAP were used to check the real dose of MAP on sponges.
The progestagen (real and residual MAP) concen-trations were determined according to Dziuk and Cook (1966). Brie¯y, MAP was extracted with chloro-form, which was then evaporated and after dilution with ethilic alcohol MAP concentrations were deter-mined by measuring the absorbances of the standards and samples by spectrophotometry at 241 nm. The amount of MAP absorbed was determined as a dif-ference between real and residual MAP.
Statistical differences for residual and absorbed MAP among groups were determined by analysis of variance. When signi®cant, comparisons between means were performed by LSD.
2.2. Experiment II
Females (n549) were randomly allocated to the following groups: Group I (n82): ewes were treated with polyurethane sponges impregnated with 40 mg MAP. Group II (n187): ewes were treated with polyurethane sponges impregnated with 50 mg MAP. Group III (n280): ewes were treated with polyurethane sponges impregnated with 60 mg MAP. Pessaries were inserted deep into the vagina and left in place for 14 days. The onset of estrus was carried out by the use of vasectomized rams ®tted with harnesses in a ratio of 1:25. The males were intro-duced in the herd after pessary removal for a total period of 96 h. Ewes were inspected for the presence of marks at 12-h intervals.
Semen from a ram with probed fertility was col-lected using an arti®cial vagina. Having determined seminal characteristics, it was diluted with milk exten-der. Some ewes from groups I (n16), II (n17) and III (n11) that had exhibited estrus were arti®cially inseminated with fresh diluted semen containing a dose of 200 millions total sperm. Cervical arti®cial insemination was performed 12 h after estrus onset. After 60 days of arti®cial insemination, pregnancy was con®rmed by ultrasound.
meanS.E.M. interval to estrus onset after treatment among groups were determined by analysis of var-iance. Pregnancy rate of the synchronized estrus for groups I, II and III were compared usingw2-test.
3. Results
Real dose of MAP on sponges are shown in Table 1. Results for Experiment I of residual MAP levels found on removed sponges are presented in Table 1. Sig-ni®cant differences (p<0.05) were found for residual MAP levels among experimental groups. MAP levels utilized by the ewes during treatment are shown in Table 1. There were no signi®cant differences for absorbed MAP levels among treatments.
In Experiment II, one ewe from Group II and three ewes from Group III lost their sponges, so they were excluded from all subsequent analysis.
Percentage of ewes that came into estrus during the controlled period and time interval between sponge removal and the onset of estrus for groups I, II and III are presented in Table 2. There were no statistical
differences for estrus incidence for females treated with the tested doses. In the same way, interval to estrus onset after synchronization treatment did not differ signi®cantly among groups.
Frequency of estrus onset for those females that were effectively synchronized is shown in Fig. 1. Regardless of almost 1% of the animals which exhib-ited estrus 24 h following sponge removal, estrus appearance for all groups occurred between 36 and 84 h after the end of treatment.
Pregnancy rate at the synchronized and inseminated estrus are shown in Table 2. No signi®cant differences were found among groups regarding this parameter.
4. Discussion
As it was demonstrated for progesterone sponges (Mac Donnell, 1985), it was observed that the pro-gestagen dose used in sponges to synchronize estrus in ewes, after 14 days treatment, was not entirely uti-lized. Results obtained in this study suggest that differences among initial doses of MAP correlate with
Table 1
Real MAP and amount of MAP remaining and absorbed in ewes treated with intravaginal sponges impregnated with different doses of MAP for 14 days
Treatment group Real MAP, meanS.E.M. (mg)
Residual MAP, meanS.E.M. (mg)
Absorbed MAP, meanS.E.M. (mg) I (40 mg MAP) 39.601.10 17.98a1.97 21.62a1.97 II (50 mg MAP) 44.101.06 24.32b2.03 19.78a2.03 III (60 mg MAP) 59.101.26 34.25c3.23 24.85a3.23
a,b,cDifferent superscripts within a column differ signi®cantly (p<0.05).
Table 2
Estrus response, time interval to onset of estrus and pregnancy rate in ewes treated with polyurethane sponges impregnated with different doses of MAPa
Treatment group
Estrus response (%)
Time to estrus onset, meanS.E.M. (h)
Pregnancy rate at the AI estrus (%)
I 79.27 55.941.87 43.75
(40 mg MAP) (65/82) (7/16)
II 77.42 56.741.13 52.94
(50 mg MAP) (144/186) (9/17)
III 80.87 57.701.02 45.45
(60 mg MAP) (224/277) (5/11)
differences among residual MAP remaining on sponges following treatment, but not with the absorbed levels. Mobarak et al. (1984) found the level of progestagen absorbed to increase as the initial dose increased. Contrary to this ®nding, Greyling et al. (1997) informed that the dose of progestagen in the sponge was super¯uous.
In relation to percentage of ewes that came into estrus during the controlled period, Robinson et al. (1968) informed the existence of a linear effect of different doses of other progestagen (Cronolone: 10, 20 and 30 mg). However, these differences were not signi®cant. Deweese et al. (1970) failed to ®nd differ-ences on estrus incidence in ewes following treatment with intravaginal sponges impregnated with 40 or 60 mg MAP. More recently, Greyling et al. (1997) reported similar results with halved sponges presum-ably containing half the standard dose of MAP. Data on percentage of ewes that came into estrus obtained in this study are coincident with these last reports.
Results show a similar time from sponge withdra-wal to estrus onset among the tested doses. In the
comprehensive study of synthetic progestagens impregnated in sponges, an extension in the time from the end of treatment to estrus appearance was sug-gested as the progestagen dose is increased and its reason could be attributed to the existence of more residual progestagen following sponge withdrawal. Supporting this hypothesis, Robinson et al. (1968) reported a signi®cant effect of different doses of other progestagen (Cronolone: 10, 20 and 30 mg) upon time interval from sponge removal to estrus onset. In their study, ewes treated with the lower dose of progestagen exhibited an earlier estrus response. Greyling et al. (1997) compared the use of whole or halved sponges and although not signi®cant, those females treated with halved sponges had a tendency to be in estrus sooner than those treated with whole sponges.
The effect of progestagen dose on fertility at the synchronized estrus has been discussed. Deweese et al. (1970), reported a tendency towards a higher percen-tage of ewes conceiving at the ®rst estrus following treatment with sponges containing 40 mg MAP in comparison with those impregnated with 60 mg MAP. More recently, Crosby et al. (1991) postulated that the presence of a high level of progestagen followed by its rapid drop is a condition to achieve acceptable fertility. This hypothesis was studied by Freitas et al. (1996) who showed that the increase of progestagen level at the end of treatment in goats did not improve estrus synchronization and can decrease fertility. Recent work performed by Greyling et al. (1997) indicated that fertility following treatment with halved pessaries was higher than that obtained with whole ones. In the present study, it was detected that pregnancy following insemination at the synchro-nized estrus was similar among the three doses employed.
In conclusion, this study suggests that the amount of MAP effectively utilized by ewes in an estrus syn-chronization treatment is lower than the dose con-ventionally used in sponges. Moreover, the absorbed levels of MAP are similar among the doses tested (40, 50 and 60 mg). Estrus incidence, interval to estrus onset and pregnancy rate were similar following treat-ment with intravaginal sponges impregnated with 40, 50 or 60 mg MAP. Therefore, it is suggested that under similar conditions, cyclic ewes could be effectively treated with a dose as low as 40 mg MAP in an estrus synchronization and arti®cial insemination program.
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